Thromboplastin reagents activate the extrinsic pathway of coagulation and are the basis for the prothrombin time (PT) test. The PT test is used to screen for blood coagulation factor deficiencies and for monitoring oral anticoagulant therapy (e.g. coumadin). Reagents for PT tests include tissue thromboplastin, also called tissue factor, and calcium ions as active ingredients diluted with appropriate buffers and stabilizers. Thromboplastin forms a complex with coagulation factor VII to greatly enhance its proteolytic activity.
Thromboplastin may be derived from a variety of tissues of different animal sources. Each tissue has a characteristic activity and sensitivity to coagulation enzymes (i.e., factors); these properties are modulated by other constituents of the reagent. Thromboplastin sensitivity is defined as the prolongation of the clotting times of both coumadinized plasmas and plasmas deficient in clotting factors II, V, VII, and X. Sensitivity to coumadinized plasma is assessed by taking the ratio of an abnormal plasma sample to a normal plasma sample.
Currently, the most sensitive thromboplastin reagents are derived from human brain and placenta. The limited availability of these materials, their cost and the potential for HIV virus contamination limit their universal acceptance. Thromboplastins derived from rabbit brain, the most common source, typically have relatively low sensitivity compared to thromboplastins derived from human tissues. The source of the thromboplastin, the method of extracting the thromboplastin and the reagent composition are all important parameters in determining reagent sensitivity. Variations in the composition of PT reagents can also be used to improve stability and adjust clotting times of plasma samples from normal individuals.
Historically thromboplastin has been extracted from tissues by heating the tissue in water or saline solutions. Thromboplatin reagents made by Baxter Healthcare Corporation, Dade Division, contain thromboplastins extracted in saline-tartrate solutions (U.S. Pat. No. 3,522,148--Jul. 25, 1970). These extracts are centrifuged to remove large particles. The supernate thromboplastin extract contains the active thromboplastin along with the sodium chloride and sodium tartrate from the extraction fluid. In Thromboplastin C the thromboplastin extract is added to a solution containing calcium lactate, sodium chloride, sodium tartrate, glycine and carboxymethyl cellulose. The final concentration of extract is 25% of the final reconstituted volume. In Thromboplastin FS, the thromboplastin extract is added to a solution containing imidazole, calcium lactate, sodium chloride, sodium tartrate, glycine and carboxymethyl cellulose. Because the final concentration of extract is 50% of the final reconstituted volume, Thromboplastin FS (TPES) made by Baxter Healthcare Corporation, Dade Division, has a relatively high concentration of rabbit brain extract. While more sensitive than other rabbit brain thromboplastins, the normal range PT values are longer than desired, the turbidity is high and the stability is less than optimal.
Boehringer Mannheim has developed a process to make rabbit brain thromboplastin more sensitive (DE 3150594A1). In their procedure, rabbit brain powder is mixed with equal parts of cellulose powder and washed with sodium acetate buffer at pH 6.5-8 to remove contaminants such as hemoglobin. The brain residue is then extracted with surface active agents, such as sodium deoxycholate in the presence of calcium ions. The key constituent disclosed in the Boehringer Mannheim process is calcium ions, and, if needed, a surface active agent is used. The use of barium sulfate is discussed in the Boehringer Mannheim patent, but discounted: "according to our own experiences, the thromboplastin largely co-precipitates with the barium sulfate." In conventional procedures the relative insensitivity of thromboplastins is due, in part, to the presence of coagulation factor VII, being tightly bound to the thromboplastin. Barium sulfate is commonly used as an adsorbing agent to remove vitamin K-dependent coagulant proteins such as factors II, VII, IX, and X.